Structured illumination apparatus, structured illumination microscopy, and structured illumination method

ABSTRACT

A structured illumination apparatus includes a light modulator being disposed in a light path of an exit light flux from a light source, and in which a sonic wave propagation path is arranged in a direction traversing the exit light flux; a driving unit generating a sonic standing wave in the sonic wave propagation path by giving a driving signal for vibrating a medium of the sonic wave propagation path to the light modulator; an illuminating optical system making at least three diffracted lights of the exit light flux passed through the sonic wave propagation path to be interfered with one another, and forming interference fringes of the diffracted lights on an observational object; and a controlling unit controlling a contrast of the interference fringes by modulating a phase of at least one diffracted light among the diffracted lights in a predetermined pitch.

CROSS-REFERENCE TO THE RELATED APPLICATION

This application is a continuation application of InternationalApplication PCT/JP2012/004548, filed Jul. 13, 2012, designating theU.S., and claims the benefit of priority from Japanese PatentApplication No. 2011-156456, filed on Jul. 15, 2011, the entire contentsof which are incorporated herein by reference.

BACKGROUND

1. Field

The present application relates to a structured illumination apparatus,a structured illumination microscopy, and a structured illuminationmethod.

2. Description of the Related Art

Patent Document 1 (U.S. Pat. No. 6,239,909) discloses an example inwhich a structured illumination microscopy is applied to a fluorescentobservation. In a method of Patent Document 1, a light flux that exitsfrom a coherent light source is split into two light fluxes by adiffraction grating, and those two light fluxes are individually focusedon mutually different positions on a pupil plane of an objective lens.The two light fluxes exit from the objective lens as collimated lightfluxes with different angles, and overlap each other on a sample planeto form interference fringes. Accordingly, the sample plane is subjectedto structured illumination. Further, in the method of Patent Document 1,images of sample images are obtained using different phase of thestructured illumination, and separated for frequency components anddemodulated from the plurality of obtained images by the methoddescribed in the U.S. Patent Document 1.

Note that as a method of shifting the phase of structured illuminationin steps, there are a method in which a wedge-shaped prism is insertedinto one of the above-described two light fluxes and moved in steps in adirection perpendicular to an optical axis, a method in which adiffraction grating is moved in steps in a direction perpendicular to agrid line, a method in which a sample is moved in steps in a pitchdirection of structured illumination, and the like.

However, when an optical element is moved in steps, a certain period oftime is required for moving and stopping of optical element at anappropriate position, so that it is difficult to reduce a period of timeto take all of the required images. Particularly, when a sample toobtain the images is a live organism specimen, there is a chance that astructure of the sample changes every second, so that the obtainment ofimages should be performed as fast as possible.

Further, as an application of technique utilizing the interferencefringes (Patent Document 1), a technique of turning a beam thatcontributes to the interference fringe into three beams (Non-PatentDocument 1: Mats G. L. Gustafsson et al., “Doubling the lateralresolution of wide-field fluorescence microscopy using structuredillumination”, Proceedings of the SPIE—The International Society forOptical Engineering, Vol. 3919, pp. 141-150, 2000) has also beenproposed for achieving a super-resolution effect in both of an in-planedirection and a depth direction of a sample. This is because, if threebeams are used, a stripe pattern of structured illumination can begenerated not only in the in-plane direction but also in the depthdirection. However, in that case, the number of images required for theaforementioned separating calculation is increased, so that it can beconsidered that the necessity of increasing the speed of obtainingimages is particularly high.

SUMMARY

The present application has been made to solve the problems of therelated art described above. A proposition of the present application isto provide a structured illumination apparatus, a structuredillumination microscopy, and a structured illumination method capable ofperforming a high-speed switching of structured pattern, and expanding astructuring direction.

One aspect of a structured illumination apparatus of the presentembodiment includes a light modulator being disposed in a light path ofan exit light flux from a light source, and in which a sonic wavepropagation path is arranged in a direction traversing the exit lightflux; a driving unit generating a sonic standing wave in the sonic wavepropagation path by giving a driving signal for vibrating a medium ofthe sonic wave propagation path to the light modulator; an illuminatingoptical system making at least three diffracted lights of the exit lightflux passed through the sonic wave propagation path to be interferedwith one another, and forming interference fringes of the diffractedlights on an observational object; and a controlling unit controlling aphase of at least one diffracted light among the diffracted lights in apredetermined pitch.

A structured illumination microscopy of the present embodiment includesthe structured illumination apparatus of the present embodiment; and animage processing unit making a super-resolved image of an observationalobject using images obtained by a detector that detects an observationallight from the observational object illuminated by the structuredillumination apparatus with a plurality of different phase of the sonicstanding wave.

One aspect of a structured illumination method of the present embodimentincludes a light modulating step preparing a light modulator beingdisposed in a light path of an exit light flux from a light source, andin which a sonic wave propagation path is arranged in a directiontraversing the exit light flux; a driving step generating a sonicstanding wave in the sonic wave propagation path by giving a drivingsignal for vibrating a medium of the sonic wave propagation path to thelight modulator; an illuminating step making at least three diffractedlights of the exit light flux passed through the sonic wave propagationpath to be interfered with one another, and forming interference fringesof the diffracted lights on an observational object; and a controllingstep controlling a phase of at least one diffracted light among thediffracted lights in a predetermined pitch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a configuration diagram of a structured illuminationmicroscopy system of an embodiment, FIG. 1B is an enlarged diagram of aperiphery of a mask 5A, FIG. 1C is a schematic diagram in which the mask5A is seen from an optical axis direction, and FIG. 1D illustrates amodified example of the mask 5A.

FIG. 2(A) is a schematic diagram illustrating a pattern of ultrasonicstanding wave generated in an ultrasonic wave propagation path R of anultrasonic wave spatial light modulator 3, FIG. 2(B) is a schematicdiagram illustrating a pattern of two-beam structured illumination(arrangement of bright part and dark part) corresponding to the pattern.FIG. 2(C) to FIG. 2(E) are diagrams explaining a change in a number offringe when a number of wave is changed.

FIG. 3(A) is a diagram explaining a relation between a length L and adistance D, FIG. 3(B) is a conceptual diagram of structured illuminationS′ corresponding to a spot S, and FIG. 3(C) is a diagram explaining adeviation of a number of fringe of the structured illumination S′.

FIG. 4 is a configuration diagram of the ultrasonic wave spatial lightmodulator 3.

FIG. 5 is a diagram explaining a basic configuration of a controllingdevice 19 (driving circuit 19A).

FIG. 6 is an operational flow chart of image processing in atwo-dimensional mode.

FIG. 7 is a diagram illustrating a time-variation of a refractive indexdistribution of an ultrasonic wave propagation path.

FIG. 8(A) is a schematic diagram illustrating a pattern of ultrasonicstanding wave, and FIG. 8(B) is a schematic diagram illustrating apattern of two-beam structured illumination.

FIG. 9(A) is a schematic diagram illustrating a pattern of ultrasonicstanding wave, and FIG. 9(B) is a schematic diagram illustrating apattern of three-beam structured illumination.

FIG. 10 is a diagram explaining a configuration of a controlling device19 related to a three-dimensional mode.

FIG. 11A is a diagram illustrating a waveform of sine signal output froma first output terminal, and FIG. 11B is a diagram illustrating awaveform of pulse signal output from a second output terminal.

FIG. 12A to FIG. 12C are diagrams each comparing a waveform oftime-variation of a refractive index of antinode a of an ultrasonicstanding wave (solid line) and a waveform of time-variation of an amountof phase modulation of 0th-order diffracted light (dotted line). FIG.12A illustrates a waveform when a phase difference ΔΨ of these twowaveforms is a product of π/2 multiplied by an even number, FIG. 12Billustrates a waveform when the phase difference ΔΨ of the two waveformsis not a product of π/2 multiplied by an odd number, and FIG. 12Cillustrates a waveform when the phase difference ΔΨ of the two waveformsis a product of π/2 multiplied by an odd number.

FIG. 13(A) illustrates a waveform of time-variation of a refractiveindex of antinode a of a standing wave in FIG. 7, FIG. 13(B) illustratesa waveform of time-variation of an amount of phase modulation of0th-order diffracted light, and FIG. 13(C) illustrates a waveformindicating a charge storage period (note that a case where N=3 isillustrated).

FIG. 14 illustrates an example of phase modulation pattern when a targetof phase modulation is 0th-order diffracted light.

FIG. 15 illustrates an example of phase modulation pattern when a targetof phase modulation is ±first-order diffracted lights.

FIG. 16A and FIG. 16B illustrate examples of mask when a target of phasemodulation is ±first-order diffracted lights.

FIG. 17 is a diagram explaining a phase shift pitch under a setting ofD:L=1:6.

FIG. 18 illustrates a modified example of the ultrasonic wave spatiallight modulator 3.

FIG. 19 illustrates another example of the mask when a target of phasemodulation is ±first-order diffracted lights.

FIG. 20 illustrates an example of phase modulation pattern when a targetof phase modulation is ±first-order diffracted lights.

DETAILED DESCRIPTION OF THE EMBODIMENT

Hereinafter, a structured illumination microscopy system will bedescribed as an embodiment of the present invention.

FIG. 1A is a configuration diagram of a structured illuminationmicroscopy system of the present embodiment.

As illustrated in FIG. 1A, in the present system, there are disposed acoherent light source 1, a collector lens 2, an ultrasonic wave spatiallight modulator 3, a lens 4, a mask 5A, a lens 6, a field stop 5B, alens 7, an excitation filter 8 a, a dichroic mirror 8, a fluorescencefilter 8 b, a tube lens 11, an imaging device (CCD camera or the like)12, a controlling device 19, an image storage/processing unit (computeror the like) 13, an image display device 14, and an objective lens 9.Note that a reference numeral 10 in FIG. 1A denotes a specimen placed ona not-illustrated stage, and in this case, it is assumed that thespecimen is previously fluorescent-stained.

The coherent light source 1 radiates light having a wavelength which isthe same as an excitation wavelength of the specimen 10. The lightexited from the coherent light source 1 is converted into collimatedlight by the collector lens 2 to be incident on the ultrasonic wavespatial light modulator 3.

The ultrasonic wave spatial light modulator 3 has an ultrasonic wavepropagation path R propagating an ultrasonic wave in a directionperpendicular to an optical axis, and gives, by generating a planarstanding wave of ultrasonic wave (referred to as “ultrasonic standingwave”, hereinafter) in the ultrasonic wave propagation path R, arefractive index distribution of sinusoidal shape to the ultrasonic wavepropagation path R. Such an ultrasonic wave spatial light modulator 3operates as a phase type diffraction grating with respect to theincident light, and branches the light into diffracted lights ofrespective orders (0th-order diffracted light, + first-order diffractedlight, − first-order diffracted light, + second-order diffracted light,− second-order diffracted light, . . . ). Note that in FIG. 1A, only0th-order diffracted light and ± first-order diffracted lights areillustrated as a representative. In FIG. 1A and FIG. 1B, a solid lineindicates the 0th-order diffracted light, and a dotted line indicatesthe ± first-order diffracted lights. The diffracted lights of respectiveorders exited from the ultrasonic wave spatial light modulator 3 passthrough the lens 4, and then form a pupil conjugate plane.

Here, the pupil conjugate plane indicates a focal position of the lens 4(rear focal position), and a position conjugated with a pupil plane P ofthe later-described objective lens 9 (position at which the ±first-order diffracted lights are condensed) via the lens 7 and the lens6 (note that a position determined by a person skilled in the art bytaking the design requirements such as aberration, vignetting and thelike of the objective lens 9 and the lenses 6 and 7, also falls into theconcept of “conjugate position”).

The mask 5A is disposed in the pupil conjugate plane, and has a functionof blocking higher-order diffracted light of second-order or higher, outof diffracted lights of respective orders which are incident on thepupil conjugate plane. Further, as illustrated in FIG. 1C in an enlargedmanner, the mask 5A is obtained by forming an optical phase modulator 5Cand a corrective block 5C′ on a substrate. Out of the above, a positionat which the optical phase modulator 5C is disposed corresponds to anarea, on the pupil conjugate plane, on which the 0th-order diffractedlight is incident, and a position at which the corrective block 5C′ isdisposed corresponds to each of areas, on the pupil conjugate plane, onwhich ± first-order diffracted lights are incident. FIG. 1C illustratesan example of disposition relationship of the optical phase modulator 5Cand the corrective block 5C′ (note that FIG. 1C is a schematic diagram,and an illustration of electrode, wiring and the like is omitted).

The optical phase modulator 5C is an optical phase modulator thatmodulates a phase of incident light (0th-order diffracted light, in thiscase) in a time direction in a modulation width π. To the optical phasemodulator 5C, an EO modulator (electro-optical modulator) that utilizesthe Kerr effect, an EO modulator that utilizes the Pockels effect, an AOmodulator (acousto-optical modulator) or the like can be applied. Theoptical phase modulator 5C is controlled by the controlling device 19.

The corrective block 5C′ is a phase block for making a phase differencebetween 0th-order diffracted light passed through the mask 5A and ±first-order diffracted lights passed through the mask 5A when an amountof phase modulation of the optical phase modulator 5C is zero (when novoltage is applied to the optical phase modulator 5C) to be zero.

Here, although details will be described later, a branching direction ofdiffracted light branched by the ultrasonic wave spatial light modulator3 can be switched. In this case, an incident area of 0th-orderdiffracted light on the pupil conjugate plane is unchanged, but, anincident area of ± first-order diffracted lights on the pupil conjugateplane moves around an optical axis. Accordingly, a position at which thecorrective block 5C′ is disposed is desirably set to an entire area onwhich ± first-order diffracted lights may be incident, as illustrated inFIG. 1D, for example. In the description hereinbelow, it is assumed thatthe position at which the corrective block 5C′ is disposed is set to theentire area on which the ± first-order diffracted lights may beincident, as illustrated in FIG. 1D.

Further, in this case, it is set that the optical phase modulator 5Calso has a function of blocking the incident light (0th-order diffractedlight, in this case) according to need (light-blocking function), inaddition to the function of modulating the phase of incident light(0th-order diffracted light, in this case) (phase-modulating function),for performing switching between a two-dimensional mode and athree-dimensional mode to be described later. Note that it is alsopossible to additionally provide a shutter that opens/closes anindependent optical path of 0th-order diffracted light, instead ofproviding the light-blocking function to the optical phase modulator 5C,but, in the description hereinbelow, it is assumed that thelight-blocking function is provided to the optical phase modulator 5C.

Incidentally, FIG. 1A illustrates a state where the light-blockingfunction of the optical phase modulator 5C is turned on, and FIG. 1Billustrates a state where the light-blocking function of the opticalphase modulator 5C is turned off.

First, explanation will be made by assuming the state where thelight-blocking function of the optical phase modulator 5C is turned on.In this case, diffracted lights that pass through the mask 5A are onlythe ± first-order diffracted lights, as illustrated in FIG. 1A.

A conjugate plane of the specimen 10 is formed by the ± first-orderdiffracted lights passed through the mask 5A via the lens 6. In thevicinity of the conjugate plane of the specimen 10, the field stop 5B isdisposed, and the field stop 5B has a function of controlling a size ofilluminated area (observational area) on the specimen 10.

The ± first-order diffracted lights passed through the field stop 5Bpass through the lens 7, and after that, the lights are incident on thedichroic mirror 8 via the excitation filter 8 a, and reflected by thedichroic mirror 8. The ± first-order diffracted lights reflected by thedichroic mirror 8 respectively form spots at mutually differentpositions on the pupil plane P of the objective lens 9. Note that theformation positions of the two spots formed by the ±first-orderdiffracted lights on the pupil plane P are at approximately an outermostperipheral portion of the pupil plane P, and positions symmetric to eachother with respect to an optical axis of the objective lens 9. In thiscase, the ± first-order diffracted lights exited from the tip of theobjective lens 9 illuminate the specimen 10 from mutually opposingdirections at an angle corresponding to NA of the objective lens 9. Notethat when a pitch of diffraction grating is changed just a little as aresult of slightly changing a frequency of applied voltage, as will bedescribed later, the positions of the two spots are extremely slightlychanged.

Here, the ± first-order diffracted lights irradiated to the specimen 10are mutually coherent lights exited from the coherent light source 1.Accordingly, by the ± first-order diffracted lights, stripedinterference fringes with a uniform fringe pitch are projected onto thespecimen 10. Specifically, an illumination pattern with respect to thespecimen 10 corresponds to an illumination pattern having a fringestructure. The illumination with the illumination pattern having thefringe structure as above is structured illumination. In the fluorescentarea (fluorescent-stained area described above) of the specimen 10subjected to the structured illumination, a fluorescent material isexcited to generate fluorescence.

Note that only two beams of the ± first-order diffracted lights are usedto realize the structured illumination, so that the illumination isstructured in an in-plane direction of the specimen 10, but, it is notstructured in a depth direction (optical axis direction) of the specimen10. Hereinafter, such structured illumination is referred to as“two-beam structured illumination”.

When the two-beam structured illumination is employed, a moiré fringecorresponding to a difference between a spatial frequency of thetwo-beam structured illumination and a spatial frequency of thefluorescent area (corresponding to a spatial frequency of the specimen)appears on the specimen 10. On the moiré fringe, a spatial frequency ofthe structure of the fluorescent area is modulated to be shifted to aspatial frequency that is lower than the actual spatial frequency.Therefore, with the use of the two-beam structured illumination, even afluorescence that exhibits a high component of spatial frequency in thestructure of the fluorescent area, namely, a fluorescence emitted at alarge angle that exceeds a resolution limit of the objective lens 9, canbe incident on the objective lens 9.

The fluorescence that is emitted from the specimen 10 and incident onthe objective lens 9 is converted into collimated light by the objectivelens 9, and then incident on the dichroic mirror 8. The fluorescencetransmits through the dichroic mirror 8, and then passes through thetube lens 11 via the fluorescence filter 8 b, to thereby form afluorescent image of the specimen 10 on an imaging plane of the imagingdevice 12. Note that this fluorescent image includes not only structuralinformation of the fluorescent area of the specimen 10 but alsostructural information of the two-beam structured illumination, and inthis fluorescent image, the spatial frequency of the structure of thefluorescent area of the specimen 10 is still being modulated (namely,the spatial frequency is still being shifted to the spatial frequencythat is lower than the actual spatial frequency).

The controlling device 19 controls the ultrasonic standing wavegenerated in the ultrasonic wave propagation path R of the ultrasonicwave spatial light modulator 3, to thereby change patterns of thetwo-beam structured illumination (details will be described later).Further, the controlling device 19 drives the imaging device 12 when thepatterns of the two-beam structured illumination are under respectivestates to obtain a plurality of types of image data, and sequentiallysends the plurality of image data to the image storage/processing unit13. Note that a charge storage time per one frame in the imaging device12 is, for example, 1/30 seconds, 1/60 seconds or the like.

The image storage/processing unit 13 performs separating calculationfrom the plurality of image data which are taken therein, to therebyobtain image data as a result of separated frequency information of thestructure of the specimen. Further, the image storage/processing unit 13performs demodulating calculation with the use of multiplication with ademodulation coefficient on the image data as a result of separatedfrequency information to obtain demodulated image data as a result ofreturning the spatial frequency of the structural information of thefluorescent area to the actual spatial frequency, and sends thedemodulated image data to the image display device 14. Note that forconcrete calculation, a method disclosed in, for example, U.S. Pat. No.8,115,806 can be employed. Accordingly, a resolved image that exceedsthe resolution limit of the objective lens 9 (two-dimensionalsuper-resolved image) is displayed on the image display device 14.

FIG. 2(A) is a schematic diagram illustrating a pattern of ultrasonicstanding wave generated in the ultrasonic wave propagation path R, andFIG. 2(B) is a schematic diagram illustrating a pattern of two-beamstructured illumination (arrangement of bright part and dark part)corresponding to the pattern (note that actually, only a pattern of areathrough which an effective light flux passes, out of the pattern of theultrasonic standing wave, contributes to the pattern of the two-beamstructured illumination). Further, in FIG. 2(A), there are two waves inultrasonic standing wave, which is smaller than the actual number, foreasier understanding of the explanation.

As illustrated in FIG. 2(A), when the number of wave of the ultrasonicstanding wave (the number of wave is counted as one when the phase isshifted by 2π) is “2”, a number of fringe (number of bright part or darkpart) of the two-beam structured illumination formed by the interferenceof ± first-order diffracted lights becomes “4”, as illustrated in FIG.2(B). Specifically, the number of fringe of the two-beam structuredillumination becomes twice the number of wave of the ultrasonic standingwave corresponding to the number of fringe.

Further, when the number of wave of the ultrasonic standing wave ischanged, by ½, in three ways such as 2, (2+½), and 3 (namely, when thewavelength of the ultrasonic standing wave is changed), as illustratedin FIG. 2(C), FIG. 2(D), and FIG. 2(E), for example, the number offringe of the two-beam structured illumination corresponding to thenumber of wave is changed, by one, in three ways such as 4, 5, and 6.

Here, if attention is focused only on a portion deviated by ½ from oneend of the ultrasonic wave propagation path R, as indicated by a whitearrow mark in FIG. 2, the phase of the two-beam structured illuminationcorresponding to the focused portion is shifted, by “π”, in three ways.

Further, if attention is focused only on portions each deviated by ⅓from one end of the ultrasonic wave propagation path R, as indicated byblack arrow marks in FIG. 2, the phase of the two-beam structuredillumination corresponding to each of the focused portions is shifted,by “2π/3”, in three ways.

Accordingly, if an incident area of light with respect to the ultrasonicwave propagation path R is tentatively limited only to the positionindicated by the white arrow mark, the phase of the two-beam structuredillumination can be shifted by “n”, only by changing the number of waveof the ultrasonic standing wave by ½.

Further, if the incident area of light with respect to the ultrasonicwave propagation path R is tentatively limited only to the positionsindicated by the black arrow marks, the phase of the two-beam structuredillumination can be shifted by “2π/3”, only by changing the number ofwave of the ultrasonic standing wave by ½.

Here, the aforementioned separating calculation for separated frequencyinformation of the specimen requires at least three pieces of image datawith different phases of the two-beam structured illumination. In thatcase, it is only required to set an amount of phase shift per one stepof the two-beam structured illumination to 2π/3, for example.

In order to generate the two-dimensional super-resolved image by thetwo-beam structured illumination, a distance D from a center of spot(effective diameter) S of light which is incident on the ultrasonic wavepropagation path R to one end of the ultrasonic wave propagation path R,may be set to one-third a length L in a propagation direction of theultrasonic wave propagation path R (D=L/3), as illustrated in FIG. 3(A).

However, when the number of wave of the ultrasonic standing wavegenerated in the ultrasonic wave propagation path R is changed by ½, thenumber of wave of the ultrasonic standing wave generated in the insideof the spot S is also deviated a little, so that the number of fringe oftwo-beam structured illumination S′ corresponding to the spot S is alsodeviated a little, as illustrated in FIG. 3(B) (note that the pattern ofwave and the pattern of fringe illustrated in FIG. 3 are illustrated ina schematic manner, and thus the number of wave and the number of fringedo not always coincide with the actual numbers).

Therefore, the length L of the ultrasonic wave propagation path R is setto be large enough, compared to a diameter φ of the spot S, so that thedeviation of the number of fringe of the two-beam structuredillumination S′ can be regarded as approximately zero.

Concretely, the length L of the ultrasonic wave propagation path R andthe diameter φ of the spot S are set to satisfy a relation of φ/L<δ,with respect to an acceptable amount δ of the deviation of the number offringe of the two-beam structured illumination S′. For example, if thedeviation of the number of fringe of the two-beam structuredillumination S′ is required to be suppressed to the number of 0.15 orless, the relational expression becomes φ/L≦0.15.

In the present embodiment, if the number of fringe is controlled at anappropriate frequency by setting that the diameter φ of the spot S is 4mm and assuming that the length L of the ultrasonic wave propagationpath R is 30 mm, the deviation of fringes at each of both ends of thetwo-beam structured illumination S′ becomes one corresponding to aboutthe number of 0.068, as illustrated in FIG. 3(C). For this reason, thedeviation of the number of fringe in the entire area of the two-beamstructured illumination S′ can be suppressed to about the number of0.068+0.068=0.13.

Note that in FIG. 3(C), a dotted line indicates an ideal pattern of thetwo-beam structured illumination S′ (pattern when the deviation of thenumber of fringe is zero), a solid line indicates an actual pattern ofthe two-beam structured illumination S′, and a deviation of the both isillustrated in an exaggerated manner for easier understanding.

Note that in the above explanation, the diameter φ of the spot Ssatisfies the relation of φ/L<δ on the ultrasonic wave propagation pathR of the ultrasonic wave spatial light modulator 3, but, it does notalways have to satisfy the relation. For example, when the ±first-orderdiffracted lights exited from the ultrasonic wave spatial lightmodulator 3 are narrowed by the field stop 5B, the length L of theultrasonic wave propagation path R, a diameter φ′ of illuminated area(observational area, field area) on the specimen plane, and an opticalpower m from the specimen plane to the ultrasonic wave spatial lightmodulator 3, are only required to be set to satisfy a relation ofφ′×m/L<5, with respect to the acceptable amount δ of the deviation ofthe number of fringe of the two-beam structured illumination S′.

FIG. 4 are diagrams specifically explaining a configuration of theultrasonic wave spatial light modulator 3. FIG. 4(A) is a diagram inwhich the ultrasonic wave spatial light modulator 3 is seen from thefront (optical axis direction), and FIG. 4(B) is a diagram in which theultrasonic wave spatial light modulator 3 is seen from the side(direction perpendicular to the optical axis).

As illustrated in FIG. 4, the ultrasonic wave spatial light modulator 3includes an acousto-optical medium 15, and the acousto-optical medium 15is set to have a prismatic columnar shape having three pairs of mutuallyopposing parallel coupled side faces. Three transducers 18 a, 18 b, and18 c are individually provided on the three pairs of coupled side faces,on one side of each of the coupled side faces, and accordingly, threeultrasonic wave propagation paths are formed in one acousto-opticalmedium 15. Hereinafter, the ultrasonic wave propagation path formedbetween a formation face of the transducer 18 a and a side face 15 aopposing the formation face is set to an “ultrasonic wave propagationpath Ra”, the ultrasonic wave propagation path formed between aformation face of the transducer 18 b and a side face 15 b opposing theformation face is set to an “ultrasonic wave propagation path Rb”, andthe ultrasonic wave propagation path formed between a formation face ofthe transducer 18 c and a side face 15 c corresponding to the formationface is set to an “ultrasonic wave propagation path Rc”.

Note that a material of the acousto-optical medium 15 is, for example, aquartz glass, a tellurite glass, a dense flint glass, a flint glass orthe like, and the three pairs of coupled side faces and two bottom facesof the acousto-optical medium are respectively polished with sufficientprecision.

Here, it is assumed that lengths L of the respective three ultrasonicwave propagation paths Ra, Rb, and Rc are common (L=30 mm). Further, thelength L satisfies the aforementioned condition with respect to thediameter φ of the spot S described above. Further, the three ultrasonicwave propagation paths Ra, Rb, and Rc intersect at angles different by60° from each other, at a position separated by L/3 from one end of eachof the paths. At a position of the intersection, a center of theabove-described spot S is positioned.

The transducer 18 a is an ultrasonic wave transducer having apiezoelectric body 16 a and two electrodes 17 a individually formed onupper and lower faces of the piezoelectric body 16 a, and is joined toone side face of the acousto-optical medium 15 via the electrode 17 abeing one of the two electrodes 17 a. When an AC voltage of highfrequency with sinusoidal shape is applied between the two electrodes 17a of the transducer 18 a, the piezoelectric body 16 a vibrates in athickness direction, resulting in that a planar ultrasonic wavereciprocates in the ultrasonic wave propagation path Ra. When thefrequency of AC voltage applied between the two electrodes 17 a is setto a specific frequency (appropriate frequency), the ultrasonic wavebecomes a standing wave, so that a distribution of sinusoidal shape isgiven to a refractive index of the ultrasonic wave propagation path,over a propagation direction of the ultrasonic wave. Accordingly, theultrasonic wave propagation path Ra becomes a phase type diffractiongrating having a phase grating perpendicular to the propagationdirection of the ultrasonic wave. Hereinafter, the propagation directionin the ultrasonic wave propagation path Ra is referred to as a “firstdirection”.

Further, the transducer 18 b, which also has the same configuration asthat of the transducer 18 a, has a piezoelectric body 16 b and twoelectrodes 17 b individually formed on upper and lower faces of thepiezoelectric body 16 b, and is joined to one side face of theacousto-optical medium 15 via the electrode 17 b being one of the twoelectrodes 17 b.

Therefore, when an AC voltage of appropriate frequency is appliedbetween the two electrodes 17 b of the transducer 18 b, a planarultrasonic wave propagates in the ultrasonic wave propagation path Rb,so that the ultrasonic wave propagation path Rb becomes a phase typediffraction grating having a phase grating perpendicular to thepropagation direction of the ultrasonic wave. Hereinafter, thepropagation direction in the ultrasonic wave propagation path Rb isreferred to as a “second direction”. This second direction makes anangle of 60° with the first direction.

Further, the transducer 18 c, which also has the same configuration asthat of the transducer 18 a, has a piezoelectric body 16 c and twoelectrodes 17 c individually formed on upper and lower faces of thepiezoelectric body 16 c, and is joined to one side face of theacousto-optical medium 15 via the electrode 17 c being one of the twoelectrodes 17 c.

Therefore, when an AC voltage of appropriate frequency is appliedbetween the two electrodes 17 c of the transducer 18 c, a planarultrasonic wave propagates in the ultrasonic wave propagation path Rc,so that the ultrasonic wave propagation path Rc becomes a phase typediffraction grating having a phase grating perpendicular to thepropagation direction of the ultrasonic wave. Hereinafter, thepropagation direction in the ultrasonic wave propagation path Rc isreferred to as a “third direction”. This third direction makes an angleof −60° with the first direction.

FIG. 5 is a diagram explaining a basic configuration of the controllingdevice 19. A reference numeral 19A in FIG. 5 denotes a driving circuit19A included in the controlling device 19, and the driving circuit 19Aincludes a high-frequency AC power source 19A-1 and a selector switch19A-2.

The high-frequency AC power source 19A-1 generates an AC voltage to besupplied to the ultrasonic wave spatial light modulator 3. A frequencyof the AC voltage is controlled to an appropriate frequency (any valuewithin a range of several tens of MHz to 100 MHz, for example), by thecontrolling device 19.

Therefore, when the amount of phase shift of the two-beam structuredillumination S′ is changed in steps, in three ways of −2π/3, 0, and+2π/3, for example, the controlling device may switch the frequency ofthe AC voltage among three ways of different appropriate frequenciesf⁻¹, f₀, and f₊₁.

For example, the appropriate frequency f₀ is set to an appropriatefrequency (80 MHz) for generating ultrasonic standing waves whose numberis 100 (the number of fringe of the two-beam structured illuminationcorresponding thereto is 200) in the ultrasonic wave propagation pathsRa, Rb, and Rc each having the length L of 30 mm. With the use of theappropriate frequency f₀, the amount of phase shift of the two-beamstructured illumination S′ is zero.

In this case, the appropriate frequency f⁻¹ becomes an appropriatefrequency (79.946 MHz) for generating ultrasonic standing waves whosenumber is (100−½) (the number of fringe of the two-beam structuredillumination corresponding thereto is 199) in the ultrasonic wavepropagation paths Ra, Rb, and Rc each having the length L of 30 mm. Withthe use of the appropriate frequency f⁻¹, the amount of phase shift ofthe two-beam structured illumination S′ becomes −2π/3.

Further, the appropriate frequency f₊₁ becomes an appropriate frequency(80.054 MHz) for generating ultrasonic standing waves whose number is(100+½) (the number of fringe of the two-beam structured illuminationcorresponding thereto is 201) in the ultrasonic wave propagation pathsRa, Rb, and Rc each having the length L of 30 mm. With the use of theappropriate frequency f₊₁, the amount of phase shift of the two-beamstructured illumination S′ becomes +2π/3.

The selector switch 19A-2 is disposed between the high-frequency ACpower source 19A-1 and the ultrasonic wave spatial light modulator 3,and can switch a connection destination on the side of the ultrasonicwave spatial light modulator 3, among the three transducers 18 a, 18 b,and 18 c of the ultrasonic wave spatial light modulator 3. Theconnection destination of the switch 19A-2 is appropriately switched bythe controlling device 19.

When the connection destination of the selector switch 19A-2 is on theside of the transducer 18 a, the AC voltage is applied between the twoelectrodes of the transducer 18 a, so that only the ultrasonic wavepropagation path Ra among the three ultrasonic wave propagation pathsRa, Rb, and Rc, becomes effective.

Further, when the connection destination of the selector switch 19A-2 ison the side of the transducer 18 b, the AC voltage is applied betweenthe two electrodes of the transducer 18 b, so that only the ultrasonicwave propagation path Rb among the three ultrasonic wave propagationpaths Ra, Rb, and Rc, becomes effective.

Further, when the connection destination of the selector switch 19A-2 ison the side of the transducer 18 c, the AC voltage is applied betweenthe two electrodes of the transducer 18 c, so that only the ultrasonicwave propagation path Rc among the three ultrasonic wave propagationpaths Ra, Rb, and Rc, becomes effective.

As above, when the effective ultrasonic wave propagation path isswitched among the three ultrasonic wave propagation paths Ra, Rb, andRc, the direction of two-beam structured illumination S′ can be switchedamong a direction corresponding to the first direction, a directioncorresponding to the second direction, and a direction corresponding tothe third direction.

Accordingly, by appropriately driving the above-described ultrasonicwave spatial light modulator 3 and controlling device 19, it is possibleto generate a detailed two-dimensional super-resolved image. Concreteexplanation will be made hereinbelow.

FIG. 6 is an operational flow chart of the controlling device.Hereinafter, respective steps will be described in order.

Step S11: The controlling device sets the connection destination of theselector switch 19A-2 to a first transducer (transducer 18 a) side, tothereby set the direction of two-beam structured illumination S′ to thedirection corresponding to the first direction.

Step S12: The controlling device sets the frequency of AC voltagegenerated by the high-frequency AC power source 19A-1 to the appropriatefrequency f⁻¹, to thereby set the amount of phase shift of the two-beamstructured illumination S′ to −2π/3.

Step S13: The controlling device drives the imaging device 12 under thisstate to obtain image data I⁻¹.

Step S14: The controlling device sets the frequency of AC voltagegenerated by the high-frequency AC power source 19A-1 to the appropriatefrequency f₀, to thereby set the amount of phase shift of the two-beamstructured illumination S′ to zero.

Step S15: The controlling device drives the imaging device 12 under thisstate to obtain image data I₀.

Step S16: The controlling device sets the frequency of AC voltagegenerated by the high-frequency AC power source 19A-1 to the appropriatefrequency f₊₁, to thereby set the amount of phase shift of the two-beamstructured illumination S′ to +2π/3.

Step S17: The controlling device drives the imaging device 12 under thisstate to obtain image data I₊₁.

Step S18: The controlling device judges whether or not the setting ofdirection of the two-beam structured illumination S′ to all of theabove-described three directions is completed, in which when the settingis not completed, the process proceeds to step S19, and when the settingis completed, the flow is terminated.

Step S19: The controlling device switches the direction of two-beamstructured illumination S′ by switching the connection destination ofthe selector switch 19A-2, and then the process proceeds to step S12.

According to the above-described flow, pieces of image data Ia⁻¹, Ia₀,and Ia₊₁ regarding the first direction, pieces of image data Ib⁻¹, Ib₀,and Ib₊₁ regarding the second direction, and pieces of image data Ic⁻¹,Ic₀, and Ic₊₁ regarding the third direction are obtained. These piecesof image data are taken into the image storage/processing unit 13.

The image storage/processing unit 13 obtains demodulated image data Ia′along the first direction from the three pieces of image data Ia⁻¹, Ia₀,and Ia₊₁ regarding the first direction, obtains demodulated image dataIb′ along the second direction from the three pieces of image data Ib⁻¹,Ib₀, and Ib₊₁ regarding the second direction, and obtains demodulatedimage data Ic′ along the third direction from the three pieces of imagedata Ic⁻¹, Ic₀, and Ic⁻⁰ along the third direction. After that, theimage storage/processing unit 13 combines the three pieces ofdemodulated image data Ia′, Ib′, and Ic′ on a wave number space, thenreturns the resultant to the real space again to obtain image data I ofsuper-resolved image along the first direction, the second direction,and the third direction, and sends the image data I to the image displaydevice 14. The super-resolved image corresponds to a two-dimensionalsuper-resolved image along the three directions in the in-planedirection of the specimen 10.

As described above, in the present system, by turning on thelight-blocking function of the optical phase modulator 5C, it ispossible to realize the structured illumination formed of the ±first-order diffracted lights (specifically, the two-beam structuredillumination).

Accordingly, in the present system, by obtaining the plurality of piecesof image data by switching the patterns of the two-beam structuredillumination, it becomes possible to generate the super-resolved imagealong the in-plane direction of the specimen 10 (specifically, thetwo-dimensional super-resolved image). Hereinafter, a mode of thepresent system for generating the two-dimensional super-resolved imageis referred to as a “two-dimensional mode”.

Further, in the present system, by turning off the light-blockingfunction of the optical phase modulator 5C, it is possible to realizestructured illumination formed of the ±first-order diffracted lights andthe 0th-order diffracted light (specifically, three-beam structuredillumination).

Accordingly, in the present system, by obtaining a plurality of piecesof image data by switching patterns of the three-beam structuredillumination, it becomes possible to generate a super-resolved imagealong the in-plane direction and the optical axis direction of thespecimen 10 (specifically, a three-dimensional super-resolved image).Hereinafter, a mode of the present system for generating thethree-dimensional super-resolved image is referred to as a“three-dimensional mode”.

Hereinafter, the three-dimensional mode will be described in detail.Note that here, only a point of difference between the three-dimensionalmode and the above-described two-dimensional mode will be described.

The three-beam structured illumination projected onto the specimen 10 inthe three-dimensional mode is formed of three beams of the ± first-orderdiffracted lights and the 0th-order diffracted light, so that theillumination is structured not only in the in-plane direction of thespecimen 10 but also in the optical axis direction of the specimen 10.Besides, a pattern of the three-beam structured illumination in thein-plane of the specimen 10 is slightly different from the pattern ofthe two-beam structured illumination in the in-plane of the specimen 10.This point will be described in detail hereinafter.

First, the ultrasonic standing wave is generated in the ultrasonic wavespatial light modulator 3 as described above, and an instantaneous valueof a pattern of the ultrasonic standing wave (=refractive indexdistribution of ultrasonic wave propagation path) is time-varied, froman upper direction to a lower direction in FIG. 7.

In FIG. 7, a time t at a moment at which the refractive indexdistribution of the ultrasonic wave propagation path becomes flat is setto t=0, and a time-variable pitch of the refractive index distributionis set to T. Incidentally, the pitch T corresponds to a reciprocal of afrequency of an AC voltage supplied to the ultrasonic wave spatial lightmodulator 3.

Hereinafter, a period of time of t=0 to T/2 is referred to as an“anterior half period of the refractive index variation”, and a periodof time of t=T/2 to T is referred to as a “last half period of therefractive index variation”. Further, as indicated by a circle mark inFIG. 7, a portion in which the refractive index is not varied in theultrasonic wave propagation path is referred as a “node”, and a portionin which the refractive index is varied is referred to as an “antinode”.

Here, if attention is focused on an antinode a in FIG. 7, it can beunderstood that a refractive index of the antinode a becomes higher thana refractive index of a node (the medium becomes dense) in the anteriorhalf period of the refractive index variation, but, the refractive indexof the antinode a becomes lower than the refractive index of the node(the medium becomes coarse) in the last half period of the refractiveindex variation.

On the other hand, if attention is focused on an antinode b adjacent tothe antinode a in FIG. 7, it can be understood that a refractive indexof the antinode b becomes lower than a refractive index of the node (themedium becomes coarse) in the anterior half period of the refractiveindex variation, but, the refractive index of the antinode b becomeshigher than the refractive index of the node (the medium becomes dense)in the last half period of the refractive index variation.

Accordingly, if the ultrasonic wave spatial light modulator 3 isregarded as a phase type diffraction grating, a phase distribution ofthe phase type diffraction grating is reversed between the anterior halfperiod and the last half period of the refractive index variation, asillustrated in FIG. 8(A) or FIG. 9(A). Specifically, the phase typediffraction grating is laterally shifted by a half grating pitch,between the anterior half period and the last half period of therefractive index variation.

Here, the two-beam structured illumination is formed of two-beaminterference fringes, so that the number of fringe on the specimen 10 ofthe two-beam structured illumination is twice the number of grating ofthe phase type diffraction grating corresponding thereto, asschematically illustrated in FIG. 8(B). Accordingly, when the phase typediffraction grating is laterally shifted as illustrated in FIG. 8(A),the pattern of the two-beam structured illumination is changed asillustrated in FIG. 8(B). Specifically, in the two-beam structuredillumination, a lateral displacement occurs by an amount correspondingto one pitch of fringe, between the anterior half period and the lasthalf period of the refractive index variation.

Therefore, when the two-beam structured illumination is subjected totime integration over a period of time which is long enough compared tothe pitch T, patterns at respective time points of the two-beamstructured illumination are emphasized, resulting in that ahigh-contrast striped image is obtained. In this case, imaging of thetwo-beam structured illumination can be realized by the imaging device12.

On the other hand, the three-beam structured illumination is formed ofthree-beam interference fringes, so that the number of fringe on thespecimen 10 of the three-beam structured illumination becomes one timethe number of grating of the phase type diffraction gratingcorresponding thereto, as schematically illustrated in FIG. 9(B).Accordingly, when the phase type diffraction grating is laterallyshifted as illustrated in FIG. 9(A), the pattern of the three-beamstructured illumination is changed as illustrated in FIG. 9(B).Specifically, in the three-beam structured illumination, a lateraldisplacement occurs by an amount corresponding to a half pitch offringe, between the anterior half period and the last half period of therefractive index variation. This fact similarly applies to therespective aspects of the three-beam structured illumination withdifferent positions of optical axis direction.

Therefore, when the three-beam structured illumination in this state issubjected to time integration over a period of time which is long enoughcompared to the pitch T, patterns at respective time points of thethree-beam structured illumination cancel each other, resulting in thata uniform image with no contrast (gray image) is obtained. In this case,imaging of the three-beam structured illumination cannot be realized bythe imaging device 12.

The reason why the optical phase modulator 5C (refer to FIG. 1A) isprovided in the present system is for solving this problem (problemregarding the reduction in contrast) in the three-dimensional mode.

FIG. 10 is a diagram explaining a configuration of a controlling device19 regarding the three-dimensional mode. As illustrated in FIG. 10, thecontrolling device 19 includes a power circuit 19C, a phase adjustingcircuit 19D and the like, in addition to the above-described AC powersource 19A-1 and selector switch 19A-2.

The power circuit 19C has a first output terminal and a second outputterminal, in which the first output terminal is a terminal for supplyingthe aforementioned AC voltage (sine signal) to the side of theultrasonic wave spatial light modulator 3, and the second outputterminal is a terminal for supplying a pulse voltage (pulse signal) tothe side of the optical phase modulator 5C.

The power circuit 19C constantly makes a frequency of the pulse signaloutput from the second output terminal coincide with a frequency of thesine signal output from the first output terminal, and when thefrequency of the sine signal is switched by the controlling device 19,the frequency of the pulse signal is also switched in a similar manner.Note that a duty ratio (ON period/pulse period) of the pulse signal isset to a previously determined ratio (which is set to ½, in this case),and is unchanged without depending on the frequency of the pulse signal.

The phase adjusting circuit 19D is interposed between the power circuit19C and the optical phase modulator 5C, and adjusts, in accordance withan instruction from the controlling device 19, a phase relationshipbetween the sine signal and the pulse signal.

FIG. 11A is a diagram illustrating a waveform of the sine signal, andFIG. 11B is a diagram illustrating a waveform of the pulse signal. Outof the above, the waveform of the sine signal defines a waveform oftime-variation of the ultrasonic standing wave in the ultrasonic wavespatial light modulator 3 (specifically, a waveform of time-variation ofrefractive index of each of the antinodes a and b), and the waveform ofthe pulse signal defines a waveform of time-variation of an amount ofphase modulation applied to the 0th-order diffracted light by theoptical phase modulator 5C.

According to the pulse signal, it is possible to set the amount of phasemodulation of the 0th-order diffracted light to 0 in one of theaforementioned anterior half period and last half period, and to set theamount of phase modulation of the 0th-order diffracted light to π in theother of the aforementioned anterior half period and last half period.In this case, it is possible to prevent the patterns at respective timepoints of the three-beam structured illumination from cancelling eachother, resulting in that the contrast of the image of the three-beamstructured illumination can be maximized.

Hereinafter, the reason why the mutual cancellation of the patterns canbe prevented, will be described in detail. For convenience ofexplanation, it is assumed that the amount of phase modulation of the0th-order diffracted light becomes π in the anterior half period, and itbecomes 0 in the last half period.

First, a pattern of the three-beam structured illumination (phasedistribution of the three-beam structured illumination) when the phasemodulation of the 0th-order diffracted light is not conducted (when themodulating function of the optical phase modulator 5C is turned off) isconsidered.

In this case, the pattern of the three-beam structured illumination isreversed between the anterior half period and the last half period asillustrated in FIG. 9(B), so that if the pattern in the anterior halfperiod is represented by the following expression (1), the pattern inthe last half period is represented by the following expression (2).

Ii(r)=I ₀+2I+4·√I ₀ ·√I·cos(k _(z) X)·cos(k _(z) Z)+2·I·cos(2k _(x)X)  (1)

Ii(r)=I ₀+2I+4·√I ₀ ·√I·cos(k _(x) X−π)·cos(k _(z) Z)+2·I·cos(2k _(x)X−2π)  (2)

Note that X indicates a position of direction corresponding to a pitchdirection of the phase type diffraction grating in the three-beamstructured illumination, Z indicates a position of the optical axisdirection of the three-beam structured illumination, I₀ indicates anintensity of the 0th-order diffracted light, I indicates an intensity ofthe ± first-order diffracted lights, and k indicates a wave number. Ineach of the expressions (1) and (2), a first term indicates an intensityof the 0th-order diffracted light, a second term indicates a sum of anintensity of the + first-order diffracted light and an intensity of the− first-order diffracted light, a third term indicates an interferenceintensity distribution between the ±first-order diffracted lights andthe 0th-order diffracted light, and a fourth term indicates aninterference intensity distribution of the mutual ± first-orderdiffracted lights. Only the third term is different between theexpression (1) and the expression (2).

Next, a pattern of the three-beam structured illumination when the phasemodulation of the 0th-order diffracted light is conducted (when themodulating function of the optical phase modulator 5C is turned on) isconsidered.

In the three-beam structured illumination of this case, since the phasemodulation is performed on the 0th-order diffracted light in amodulation width π, the phase of the 0th-order diffracted light isshifted by it in the anterior half period, and thus the pattern in theanterior half period becomes one as represented by an expression (1′),and the pattern in the last half period is the same pattern asrepresented by the expression (2).

Ii(r)=I ₀+2I+4·√I ₀ ·√I·cos(k _(z) Xπ)·cos(k _(z) Z)+2·I·cos(2k _(x)X)  (1′)

Specifically, the three-beam structured illumination when the phasemodulation of the 0th-order diffracted light is conducted, takes thepattern of the expression (1′) in the anterior half period, and takesthe pattern of the expression (2) in the last half period. When thisexpression (1′) is compared with the expression (2), it can beunderstood that the both patterns are mutually the same.

Accordingly, when the phase modulation of the 0th-order diffracted lightis conducted, the mutual cancellation of the patterns can be prevented.

Note that here, the timing at which the phase of the 0th-orderdiffracted light is shifted is set only to the anterior half period,but, it goes without saying that a similar effect can be achieved alsowhen the timing is set only to the last half period.

Now, in advance of the three-dimensional mode, the controlling device 19continuously drives each of the coherent light source 1, the imagingdevice 12, and the phase adjusting circuit 19D in a state where auniform test specimen, in place of the specimen 10, is disposed in thepresent system, changes an amount of phase modulation of the lightintensity modulator 30 while referring to a contrast of an image outputfrom the imaging device 12, and fixes the amount of phase modulation ata time point at which the contrast becomes maximum (or at a time pointat which the contrast reaches a value equal to or greater than athreshold value).

FIG. 12 are diagrams each comparing a waveform of time-variation of therefractive index of an antinode a of the ultrasonic standing wave and awaveform of time-variation of an amount of phase modulation of the0th-order diffracted light. In FIG. 12, the waveform of time-variationof the refractive index of the antinode a is indicated by a solid line,and the waveform of time-variation of the amount of phase modulation ofthe 0th-order diffracted light is indicated by a dotted line.

As illustrated in FIG. 12A, when a phase difference ΔΨ between thewaveform of time-variation of the refractive index of the antinode a andthe waveform of time-variation of the amount of phase modulation of the0th-order diffracted light coincides with a product of π/2 multiplied byan even number, the period of time during which the phase of the0th-order diffracted light is shifted coincides with either the anteriorhalf period or the last half period of the refractive index variation,so that the contrast of the image of the three-beam structuredillumination becomes maximum.

Further, as illustrated in FIG. 12B, even if the phase difference ΔΨbetween the waveform of time-variation of the refractive index of theantinode a and the waveform of time-variation of the amount of phasemodulation of the 0th-order diffracted light does not coincide with theproduct of π/2 multiplied by an even number, the image of the three-beamstructured illumination has a contrast, as long as the phase differenceΔΨ does not coincide with a product of π/2 multiplied by an odd number.

However, as illustrated in FIG. 12C, when the phase difference ΔΨbetween the waveform of time-variation of the refractive index of theantinode a and the waveform of time-variation of the amount of phasemodulation of the 0th-order diffracted light coincides with the productof π/2 multiplied by an odd number, the contrast of the image of thethree-beam structured illumination becomes zero.

Therefore, in the three-dimensional mode, the phase difference ΔΨbetween the waveform of time-variation of the refractive index of theantinode a and the waveform of time-variation of the amount of phasemodulation of the 0th-order diffracted light is set to a product otherthan the product of π/2 multiplied by an odd number, and is desirablyset to the product of π/2 multiplied by an even number.

Note that although the description is made here by focusing attention onthe refractive index of the antinode a, the waveform of time-variationof the refractive index of the antinode b corresponds to the waveform oftime-variation of the refractive index of the antinode a in which thephase is made to be opposite, so that the same also applies to a casewhere attention is focused on the refractive index of the antinode b.

Further, similar to the controlling device in the two-dimensional mode(refer to FIG. 6), the controlling device in the three-dimensional modeswitches a connection destination of the selector switch 19A-2, tothereby switch a direction of the three-beam structured illuminationamong a first direction, a second direction, and a third direction.

Further, similar to the controlling device in the two-dimensional mode(refer to FIG. 6), the controlling device in the three-dimensional moderepeatedly obtains pieces of image data while shifting the phase of thethree-beam structured illumination in respective cases where thedirection of the three-beam structured illumination is the firstdirection, the second direction, and the third direction.

Note that in the three-dimensional mode, at least five pieces of imagedata with different phases of the three-beam structured illumination arerequired for the separating calculation for separated information ofspecimen (incidentally, it is sufficient if there are provided at leastthree pieces of image data in the two-dimensional mode).

For this reason, it is desirable that the controlling device in thethree-dimensional mode sets the amount of phase shift per one step inthe three-beam structured illumination to 2π/5, for example.

In that case, it is only required to set a distance D from a center ofspot (effective diameter) S of light which is incident on the ultrasonicwave propagation path R to one end of the ultrasonic wave propagationpath R, to one-fifth a length L in a propagation direction of theultrasonic wave propagation path R (D=L/5).

Further, the controlling device in the three-dimensional mode obtainsfive pieces of image data having amounts of phase shift which aredifferent by 2π/5, with respect to the respective directions, and sendsthe five pieces of image data regarding the first direction, the fivepieces of image data regarding the second direction, and the five piecesof image data regarding the third direction to the imagestorage/processing unit 13.

Further, the image storage/processing unit 13 in the three-dimensionalmode obtains demodulated image data along the first direction and theoptical axis direction from the five pieces of image data regarding thefirst direction, obtains demodulated image data along the seconddirection and the optical axis direction from the five pieces of imagedata regarding the second direction, and obtains demodulated image dataalong the third direction and the optical axis direction from the fivepieces of image data regarding the third direction. After that, theimage storage/processing unit 13 combines the three pieces ofdemodulated image data on a wave number space, then returns theresultant to the real space again to obtain image data of super-resolvedimage along the first direction, the second direction, the thirddirection, and the optical axis direction, and sends the image data tothe image display device 14. The super-resolved image corresponds to asuper-resolved image along the three directions in the in-plane of thespecimen 10 and the optical axis direction of the specimen 10(three-dimensional super-resolved image) (the above is the explanationof the three-dimensional mode). For the concrete calculation, a methoddisclosed in U.S. Pat. No. 8,115,806, for example, can be employed.

As described above, in the present system, the length L of theultrasonic wave propagation path R, the diameter φ of the spot S, andthe distance D from one end of the ultrasonic wave propagation path R tothe center of the spot S, are set to satisfy the optimum relationdescribed above, so that the phase of the two-beam structuredillumination or the three-beam structured illumination can be switchedonly by electrically switching the frequency of AC voltage given to theultrasonic wave spatial light modulator 3. A period of time required forthe switching is short, and can be reduced to 10 ms or less evenincluding a time constant of the circuit system including the powersource.

Therefore, a period of time for obtaining the required number of imagedata in the present system can be particularly reduced to a short periodof time, compared to a case that the optical element or the specimen 10is mechanically moved for switching the phase of the two-beam structuredillumination or the three-beam structured illumination.

Further, in the present system, there is no need to mechanically movethe optical element or the specimen 10 for switching the phase of thetwo-beam structured illumination or the three-beam structuredillumination, so that the configuration of the periphery of the opticalsystem can be simplified.

Further, in the present system, the three ultrasonic wave propagationpaths Ra, Rb, and Rc with different angles are formed in oneacousto-optical medium 15, so that the direction of the two-beamstructured illumination or the three-beam structured illumination can beswitched only by electrically changing the connection state of theselector switch 19A-2. A period of time required for the switching isshort, and can be reduced to 10 ms or less even including a timeconstant of the circuit system including the power source.

Therefore, the present system can particularly reduce the period of timefor obtaining the required number of image data, compared to a casewhich the optical element or the specimen 10 is mechanically rotated forswitching the direction of the two-beam structured illumination or thethree-beam structured illumination.

Further, in the present system, since the switching can be made betweenthe two-dimensional mode and the three-dimensional mode, it is possibleto generate both of the two-dimensional super-resolved image and thethree-dimensional super-resolved image from the same specimen 10.

Further, in the three-dimensional mode of the present system, since theultrasonic wave spatial light modulator is used as the phase typediffraction grating for generating the three-beam structuredillumination, there is a possibility that the contrast of the image ofthe three-beam structured illumination becomes zero (a possibility thatthe imaging cannot be realized). However, the optical phase modulator 5Cis used and the phase of the 0th-order diffracted light is modulated bythe pulse signal whose frequency is the same as that of the sine signal,so that such a problem (problem regarding the reduction in contrast) canbe avoided.

[Supplement Regarding Control of Contrast of Image]

Further, in the three-dimensional mode of the present system, theadjustment of phase difference ΔΨ between the waveform of time-variationof the refractive index and the waveform of time-variation of the amountof phase modulation of the 0th-order diffracted light is automaticallyconducted by the controlling device 19, but, the adjustment may also beconducted manually by a user of the system. However, in that case, it isrequired that the controlling device 19 displays, in real time, an imageoutput by the imaging device 12, to the image display device 14, so thatthe user can check the contrast of image which is under adjustment.

Further, in the three-dimensional mode of the present system, thefrequency of the pulse signal is made to coincide with the frequency ofthe sine signal, but, there is no problem if the frequency of the pulsesignal does not coincide with the frequency of the sine signal as longas the pulse signal is synchronized with the sine signal.

For example, it is also possible that the frequency of the pulse signalis set to 1/N times the frequency of the sine signal (N is an integer of1 or more), and the duty ratio (ON period/pulse period) of the pulsesignal is set to 1/(2N). FIG. 13 illustrate an example of case where itis set that N=3, in which FIG. 13(A) illustrates a waveform oftime-variation of the refractive index of the antinode a in FIG. 7 ofthe ultrasonic standing wave under the setting of N=3, and FIG. 13(B)illustrates a waveform of time-variation of the phase of the 0th-orderdiffracted light under the setting of N=3.

In this case, although the phase of the 0th-order diffracted light isshifted only once every three pitches (3T) of the refractive indexvariation, the length of the period of time during which the phase ofthe 0th-order diffracted light is shifted corresponds to a length ofhalf pitch (T/2) of the refractive index variation, so that by adjustingthe phase difference ΔΨ between the waveform of time-variation of therefractive index and the waveform of time-variation of the amount ofphase modulation of the 0th-order diffracted light to an appropriatevalue, and making the period of time during which the phase of the0th-order diffracted light is shifted coincide with only either theanterior half period or the last half period of the refractive indexvariation as illustrated in FIG. 13(A) and FIG. 13(B), the contrast ofimage can be increased.

Note that when N is set to 2 or more, the period of time in which thecontrast of image is increased becomes, not continuous, but intermittentperiod of time (the period of time in which the contrast of image isincreased appears only intermittently), so that it is also possible tolimit the charge storage period of the imaging device 12 only to theperiod of time in which the contrast of image is increased, by making adriving signal of the imaging device 12 (a signal of regulating anopening/closing timing of electronic shutter) to be synchronized withthe aforementioned sine signal.

In this case, the charge storage period may be set to a period of time(T) as a result of combining a period of time during which the phase ofthe 0th-order diffracted light is shifted (T/2) and a similar period oftime before or after the period of time (T/2), out of a phase-modulationpitch (N×T), as illustrated in FIG. 13(C), for example.

Note that here, although the duty ratio (ON period/pulse period) of thepulse signal is set to coincide with 1/(2N), if the contrast of imagemay be lowered to some degree, the duty ratio does not always have toperfectly coincide with 1/(2N), and it may be set to less than 1/(2N).

Incidentally, when the duty ratio is set to less than 1/(2N), the lengthof period of time during which the phase of the 0th-order diffractedlight is shifted becomes shorter than the length of half pitch (T/2) ofthe refractive index variation, so that it is desirable to adjust thephase difference ΔΨ between the waveform of time-variation of therefractive index and the waveform of time-variation of the amount ofphase modulation of the 0th-order diffracted light to an appropriatevalue to make a timing at which the phase of the 0th-order diffractedlight is shifted coincide with a timing at which the refractive indexindicates a peak or a valley.

Further, in the three-dimensional mode of the present system, thewaveform of time-modulation of the phase of the 0th-order diffractedlight is set to the pulse waveform, and the phase of the 0th-orderdiffracted light is shifted (the phase is switched) rapidly between 0and π, but, a certain effect can be achieved only by setting thewaveform of time-modulation to a sinusoidal waveform and gently shiftingthe phase between 0 and it.

Further, in the three-dimensional mode of the present system, thewaveform of time-variation of the phase of the 0th-order diffractedlight is set to the pulse waveform or the sinusoidal waveform, but, itis also possible to employ another waveform as long as the phase of the0th-order diffracted light is time-modulated.

Further, in the three-dimensional mode of the present system, theposition at which the mask 5A is inserted is set to the pupil conjugateplane of the light path from the ultrasonic wave spatial light modulator3 to the fluorescence filter 8 b, but, the position may also be deviatedto some degree from the pupil conjugate plane. However, when theposition is close to the pupil conjugate plane, an interval between theincident position of the 0th-order diffracted light and the incidentposition of the ± first-order diffracted lights is increased, so thatthere is an advantage that the degree of freedom of layout of theoptical phase modulator 5C and the corrective block 5C′ is increased.

Further, in the three-dimensional mode of the present system, the targetof phase modulation is set to the 0th-order diffracted light, but, itmay also be set to the ± first-order diffracted lights. FIG. 14illustrates an example of phase modulation pattern when the target ofphase modulation is the 0th-order diffracted light, and meanwhile, FIG.15 illustrates an example of phase modulation pattern when the target ofphase modulation is the ±first-order diffracted lights.

Further, when the target of phase modulation is set to the ± first-orderdiffracted lights, a position at which the optical phase modulator 5C isdisposed corresponds to each of areas on which the ± first-orderdiffracted lights are incident (two areas symmetric to each other withrespect to the optical axis), and a position at which the correctiveblock 5C′ is disposed corresponds to an area on which the 0th-orderdiffracted light is incident (area in the vicinity of the optical axis),as illustrated in FIG. 16(A). Note that in that case, a shutter foropening/closing the light path of the 0th-order diffracted light isadditionally required.

Further, when the target of phase modulation is set to the ± first-orderdiffracted lights, in order to deal with the switching of branchingdirection of diffracted light, it is only required that the entire mask5A is set to be able to rotate around the optical axis as indicated byan arrow mark in FIG. 16(A), or the position at which the optical phasemodulator 5C is disposed is set to the entire area on which the ±first-order diffracted lights may be incident, as illustrated in FIG.16(B), for example (note that FIG. 16(A) and FIG. 16(B) are schematicdiagrams, and an illustration of electrode, wiring, and the like isomitted).

Further, in the three-dimensional mode of the present system, the targetof phase modulation is set to either the ± first-order diffracted lightsor the 0th-order diffracted light for modulating the phase differencebetween the ± first-order diffracted lights and the 0th-order diffractedlight, but, it goes without saying that the target can be set to both ofthe ± first-order diffracted lights and the 0th-order diffracted light.In that case, it is only required to individually dispose the opticalphase modulator 5C in both of the area on which the ± first-orderdiffracted lights may be incident and the area on which the 0th-orderdiffracted light may be incident.

Note that in the present system, the optical phase modulator 5C is usedfor modulating the phase of the diffracted light, but, the presentinvention is not limited to this. For example, the use of refractivemember with different thicknesses, instead of the optical phasemodulator 5C, is also within expectations, and as the refractive memberwith different thicknesses, a wedge-shaped or stepped glass member orthe like can be cited. It is also possible that the wedge-shaped orstepped glass member is disposed in the area on which the 0th-orderdiffracted light is incident and the glass member is moved in apredetermined direction, or the glass member is rotated around apredetermined axis to periodically change the thickness of the glassmember through which the 0th-order diffracted light passes, to therebymodulate the phase of the diffracted light that passes through the glassmember.

[Supplement Regarding Phase Shift of Structured Illumination]

Note that in the two-dimensional mode of the present system, the changepattern of frequency of the AC voltage given to the transducer is set toa pattern in which the number of wave of the ultrasonic standing wave ischanged by ½, and the distance D from the center of the spot (effectivediameter) S to one end of the ultrasonic wave propagation path is set toone-third the length L in the propagation direction of the ultrasonicwave propagation path R (D=L/3) in order to set the phase shift pitch ofthe two-beam structured illumination in each of the first direction, thesecond direction and the third direction to 2π/3, but, the presentinvention is not limited to this.

Concretely, the ultrasonic wave propagation path is only required tosatisfy the following conditions.

First, the change pattern of frequency of the AC voltage given to thetransducer is only required to be a pattern in which the number of waveof the ultrasonic standing wave is changed by M/2 (where |M| is aninteger of 1 or more).

In that case, in order to set the phase shift pitch of the two-beamstructured illumination to an arbitrary value Δψ, the distance D fromone end of the ultrasonic wave propagation path to the center of thespot S, and the total length L of the ultrasonic wave propagation pathare only required to satisfy a relation of D:L=Δψ/M:2π.

Incidentally, if it is set that M=1, the number of ultrasonic standingwave is changed only by ½, so that the deviation occurred, due to thechange, in the number of fringe of the two-beam structured illuminationcan be minimized.

Further, when the number of image data used for the separatingcalculation is k, if it is set that Δψ=2π/k, it is possible to securelyobtain the required image data. Note that it is preferable that |k| isan integer of 3 or more.

Further, in the three-dimensional mode of the present system, the changepattern of frequency of the AC voltage given to the transducer is set toa pattern in which the number of wave of the ultrasonic standing wave ischanged by ½, and the distance D from the center of the spot (effectivediameter) S to one end of the ultrasonic wave propagation path is set toone-fifth the length L in the propagation direction of the ultrasonicwave propagation path R (D=L/5) in order to set the phase shift pitch ofthe three-beam structured illumination in each of the first direction,the second direction, and the third direction to 2π/5, but, the presentinvention is not limited to this.

Concretely, the ultrasonic wave propagation path is only required tosatisfy the following conditions.

First, the change pattern of frequency of the AC voltage given to thetransducer is only required to be a pattern in which the number of waveof the ultrasonic standing wave is changed by M/2 (where |M| is aninteger of 1 or more).

In that case, in order to set the phase shift pitch of the three-beamstructured illumination to an arbitrary value Δψ, the distance D fromone end of the ultrasonic wave propagation path to the center of thespot S, and the total length L of the ultrasonic wave propagation pathare only required to satisfy a relation of D:L=Δψ/M:2π.

Note that a passing area of exit light flux (spot) on the ultrasonicwave propagation path R of the ultrasonic wave spatial light modulator 3does not always have to be limited to a partial area separated from bothends of the ultrasonic wave propagation path R, for forming theinterference fringes on the specimen plane, and when, for example, thelight flux passed through the ultrasonic wave propagation path R isnarrowed by the field stop 5B, the passing area of effective exit lightflux on the ultrasonic wave propagation path R, namely, the partial areaon the ultrasonic wave propagation path R through which the exit lightflux that contributes to the interference fringes (structuredillumination S′) formed on the illuminated area (observational area,field area) on the specimen plane passes, is only required to satisfythe relation of D:L=Δψ/M:2π.

Incidentally, if it is set that M=1, the number of ultrasonic standingwave is changed only by ½, so that the deviation occurred, due to thechange, in the number of fringe of the three-beam structuredillumination can be minimized.

Further, when the number of image data used for the separatingcalculation is k, if it is set that Δψ=2π/k, it is possible to securelyobtain the required image data. Note that it is preferable that |k| isan integer of 5 or more.

Further, in the above explanation, the position of spot in theultrasonic wave spatial light modulator 3 is made to be differentbetween the two-dimensional mode and the three-dimensional mode, and inthis case, there is generated a necessity of moving the position of theultrasonic wave spatial light modulator 3 before and after the switchingof mode.

Accordingly, in the present system, it is preferable to previously set aswitching pattern of the sine signal described above to enable thenumber of wave of the ultrasonic standing wave to be changed by ½, andto previously adjust the positional relationship between the spot S andthe ultrasonic wave spatial light modulator 3 so that the distance Dfrom one end of the ultrasonic wave propagation path to the center ofthe spot S satisfies D:L=1:6.

In this case, in the two-dimensional mode, by changing the number ofwave of the ultrasonic standing wave by ½, it is possible to set thephase shift pitch of the two-beam structured illumination to “2π/3”.

Further, in this case, in the three-dimensional mode, by changing thenumber of wave of the ultrasonic standing wave by 1, it is possible toset the phase shift pitch of the three-beam structured illumination to“π/3”.

FIG. 17 illustrates a relation between the number of wave of theultrasonic standing wave and the amount of phase shift when it is setthat D:L=1:6.

Therefore, it is only required that the image storage/processing unit 13in the two-dimensional mode uses three pieces of image data obtained ineach of three states indicated by reference letters a, c, and e in FIG.17 for the above-described separating calculation.

Meanwhile, the image storage/processing unit 13 in the three-dimensionalmode is only required to use six pieces of image data obtained in eachof six states indicated by reference letters a to f in FIG. 17 for theabove-described separating calculation.

Specifically, in the present system, only by previously adjusting thepositional relationship between the spot S and the ultrasonic wavespatial light modulator 3 to the relationship suitable for both of thetwo-dimensional mode and the three-dimensional mode, it is possible toomit the movement of the ultrasonic wave spatial light modulator 3before and after the switching of mode.

Further, in the acousto-optical medium 15 of the present system, thethree ultrasonic wave propagation paths Ra, Rb, and Rc are disposed inan asymmetric relation relative to the center of the spot S (refer toFIG. 4), but, they may also be disposed in a symmetric relation asillustrated in FIG. 18, for example. Incidentally, an advantage of theexample illustrated in FIG. 4 is that projections and depressions of theouter shape of the acousto-optical medium 15 are small, and an advantageof the example illustrated in FIG. 18 is that environments of the threeultrasonic wave propagation paths Ra, Rb, and Rc completely coincidewith one another.

Further, in the present system, the lengths of the ultrasonic wavepropagation paths Ra, Rb, and Rc are set to common, and change patternsof frequency of the AC voltage given to the transducers 18 a, 18 b, and18 c are set to common, but, it is also possible to set that therespective lengths and change patterns are not common. However, also inthat case, the respective ultrasonic wave propagation paths Ra, Rb, andRc are set to satisfy the above-described conditions.

Further, in the explanation of the present embodiment, it is explainedthat the number of wave of the ultrasonic standing wave (namely, thewavelength of the ultrasonic standing wave) generated in the ultrasonicwave propagation paths Ra, Rb, and Rc, is changed in the predeterminedpattern for shifting the phase of the interference fringes formed of thediffracted lights, and the frequency of AC voltage given to thetransducers 18 a, 18 b, and 18 c of the ultrasonic wave spatial lightmodulator 3 is changed, as one method of changing the wavelength of theultrasonic standing wave, but, it goes without saying that the presentinvention is not limited to this method.

For example, when the optical phase modulator 5C is individuallydisposed in each of the incident area of the + first-order diffractedlight and the incident area of the − first-order diffracted light on themask 5A for modulating the phase difference between the ± first-orderdiffracted lights and the 0th-order diffracted light in thethree-dimensional mode (for example, when the optical phase modulators5C and the corrective block 5C′ are disposed as illustrated in FIG. 19),it is also possible to design as follows.

First, in the two-dimensional mode, the 0th-order diffracted light isblocked, a phase offset α is given to the + first-order diffractedlight, and a phase offset −α is given to the − first-order diffractedlight (specifically, the phase offset of the + first-order diffractedlight with respect to the 0th-order diffracted light and the phaseoffset of the − first-order diffracted light with respect to the0th-order diffracted light are set to equivalent with opposite signs).Also in this state, it is possible to generate appropriate two-beamstructured illumination. Further, when switching the phase of thetwo-beam structured illumination, the phase offset amount α is switchedin a predetermined pattern, instead of switching the wavelength of theaforementioned ultrasonic standing wave in the predetermined pattern.For example, if the phase offset amount α is switched in three ways in apitch of 2π/3, the phase of the two-beam structured illumination can beswitched in three ways in a pitch of 2π/3.

Meanwhile, in the three-dimensional mode, the 0th-order diffracted lightis transmitted, the phase offset α is given to the + first-orderdiffracted light, the phase offset −α is given to the − first-orderdiffracted light, and from that state, the aforementioned modulation isconducted. Also in this state, it is possible to generate appropriatethree-beam structured illumination (FIG. 20 is a diagram illustrating aphase modulation pattern in this case). Further, when switching thephase of the three-beam structured illumination, the phase offset amountα is switched in a predetermined pattern, instead of switching thewavelength of the aforementioned ultrasonic standing wave in thepredetermined pattern. For example, if the phase offset amount α isswitched in five ways in a pitch of 2π/5, the phase of the three-beamstructured illumination can be switched in five ways in a pitch of 2π/5.

Note that here, the variation pitch of the phase offset amount α in thetwo-dimensional mode and the variation pitch of the phase offset amountα in the three-dimensional mode are individually set, but, they may alsobe set in a common manner, Specifically, it is also possible that thesystem is previously configured so that the phase offset amount α can beswitched by 2π/6, in which three pieces of image data obtained in eachof three states where the phase offset amounts α are 0, 2(2π)/6, and4(2π)/6, are used for the above-described separating calculation in thetwo-dimensional mode, and six pieces of image data obtained in each ofsix states where the phase offset amounts α are 0, (2π)/6, 2(2π)/6,3(2π)/6, 4(2π)/6, and 5(2π)/6, are used for the above-describedseparating calculation in the three-dimensional mode.

Further, in the above-described embodiment, as the diffracted lights forforming the interference fringes (two-beam interference fringes,three-beam interference fringes), a combination of ± first-orderdiffracted lights and 0th-order diffracted light is employed, but,another combination may also be employed. In order to form thethree-beam interference fringes, it is only required to generatethree-beam interference caused by three diffracted lights in which aninterval of orders of diffraction is equal, so that a combination of0th-order diffracted light, first-order diffracted light, andsecond-order diffracted light, a combination of ±second-order diffractedlights and 0th-order diffracted light, a combination of ±third-orderdiffracted lights and 0th-order diffracted light, and the like can beemployed.

Note that all documents hereinbelow disclosed in the presentspecification are incorporated by reference.

-   1) U.S. Pat. No. 6,239,909-   2) U.S. Pat. No. 8,115,806-   3) Mats G. L. Gustafsson et al., “Doubling the lateral resolution of    wide-field fluorescence microscopy using structured illumination”,    Proceedings of the SPIE—The international Society for Optical    Engineering, Vol. 3919, pp. 141-150, 2000

The many features and advantages of the embodiments are apparent fromthe detailed specification and, thus, it is intended by the appendedclaims to cover all such features and advantages of the embodiments thatfall within the true spirit and scope thereof. Further, since numerousmodifications and changes will readily occur to those skilled in theart, it is not desired to limit the inventive embodiments to the exactconstruction and operation illustrated and described, and accordinglyall suitable modifications and equivalents may be restored to, fallingwithin the scope thereof.

What is claimed is:
 1. A structured illumination apparatus, comprising:a light modulator being disposed in a light path of an exit light fluxfrom a light source, and in which a sonic wave propagation path isarranged in a direction traversing the exit light flux; a driving unitgenerating a sonic standing wave in the sonic wave propagation path bygiving a driving signal for vibrating a medium of the sonic wavepropagation path to the light modulator; an illuminating optical systemmaking at least three diffracted lights of the exit light flux passedthrough the sonic wave propagation path to be interfered with oneanother, and forming interference fringes of the diffracted lights on anobservational object; and a controlling unit controlling a phase of atleast one diffracted light among the diffracted lights in apredetermined pitch.
 2. The structured illumination apparatus accordingto claim 1, wherein the controlling unit controls the phase in order fora phase difference between at least two diffracted lights among thediffracted lights to be π on the observational object.
 3. The structuredillumination apparatus according to claim 1, wherein the controllingunit modulates the phase of the diffracted light by a modulating signal.4. The structured illumination apparatus according to claim 3, whereinthe controlling unit sets a frequency of the modulating signal to thefrequency which is 1/N times a frequency of the driving signal and setsa duty ratio of the modulating signal to 1/(2N) or less (where N is aninteger of 1 or more).
 5. The structured illumination apparatusaccording to claim 3, wherein the controlling unit includes a phasemodulator being disposed in a light path of at least one diffractedlight among the diffracted lights, and a driving part driving the phasemodulator by the modulating signal.
 6. The structured illuminationapparatus according to claim 1, wherein the diffracted lights are an0th-order diffracted light and + first-order diffracted lights.
 7. Thestructured illumination apparatus according to claim 6, furthercomprising an offset unit offsetting a phase of the + first-orderdiffracted light relative to the 0th-order diffracted light by an offsetamount α and offsetting a phase of the − first-order diffracted lightrelative to the 0th-order diffracted light by an offset amount −α,wherein the controlling unit controls a contrast of the interferencefringes by performing the controlling from a state where the phases ofthe − first-order diffracted lights are offset, and switch a phase ofthe interference fringes by switching the offset amount α in apredetermined pattern.
 8. The structured illumination apparatusaccording to claim 1, wherein: the interference fringes are formed ofthe exit light flux being passed through a predetermined partial areaseparated from both ends of the sonic wave propagation path; and thedriving unit switches a phase of the interference fringes by switching awavelength of the sonic standing wave in a predetermined pattern.
 9. Thestructured illumination apparatus according to claim 8, wherein: thedriving unit is capable of changing the wavelength of the sonic standingwave in a pattern in which a total number of wave of the sonic standingwave is changed by M/2 (where |M| is an integer of 1 or more); and whena phase shift pitch of the interference fringes is set to Δψ, a distanceD from either end portion of the sonic wave propagation path to thepartial area and a total length L of the sonic wave propagation pathsatisfy a relation of D:L=Δψ/M:2π.
 10. The structured illuminationapparatus according to claim 8, wherein the driving unit switches thewavelength of the sonic standing wave by switching a frequency of thedriving signal given to the light modulator in a predetermined pattern.11. The structured illumination apparatus according to claim 1, furthercomprising a switching unit switching a mode of the structuredillumination apparatus between a three-dimensional mode in which threediffracted lights of the exit light flux are reflected on theinterference fringes and a two-dimensional mode in which only twodiffracted lights of the exit light flux are reflected on theinterference fringes.
 12. The structured illumination apparatusaccording to claim 11, wherein: the interference fringes are formed ofthe exit light flux being passed through a predetermined partial areaseparated from both ends of the sonic wave propagation path; the drivingunit switches a phase of the interference fringes by switching awavelength of the sonic standing wave in a predetermined pattern; and adistance D from either end portion of the sonic wave propagation path tothe partial area and a total length L of the sonic wave propagation pathsatisfy a relation of D:L=1:6.
 13. The structured illumination apparatusaccording to claim 1, wherein the light modulator has a plurality of thesonic wave propagation paths which intersect at a passing area of theexit light flux, and a direction of the sonic standing wave is capableof switching by switching an effective sonic wave propagation path amongthe sonic wave propagation paths.
 14. A structured illuminationmicroscopy, comprising: the structured illumination apparatus accordingto claim 1; and an image processing unit making a super-resolved imageof an observational object using images being obtained by a detectorthat detects an observational light from the observational objectilluminated by the structured illumination apparatus under a pluralityof states in which patterns of the sonic standing wave are different.15. A structured illumination method, comprising: preparing a lightmodulator being disposed in a light path of an exit light flux from alight source, and in which a sonic wave propagation path is arranged ina direction traversing the exit light flux; generating a sonic standingwave in the sonic wave propagation path by giving a driving signal forvibrating a medium of the sonic wave propagation path to the lightmodulator; making at least three diffracted lights of the exit lightflux passed through the sonic wave propagation path to be interferedwith one another, and forming interference fringes of the diffractedlights on an observational object; and controlling a phase of at leastone diffracted light among the diffracted lights in a predeterminedpitch.